PROJECT SUMMARY The long-term goal of the proposed project is to identify the first chemical probes and leads for developing the first clinical candidates targeting the alternative lengthening of telomeres (ALT) pathway. Cellular immortality is essential for tumorigenesis and is achieved by the action of telomerase in ~85% of cancers and the ALT mechanism in ~15% of cancers. ALT is poorly understood, but most ALT cells appear to share a common biomarker, extrachromosomal C-circles, that are predicted to be involved in telomere maintenance. In many cancers, the ALT phenotype is associated with high grade and poor prognosis. This suggests that targeting the ALT pathway will have significant impact in a substantial number of disease states. In addition to the clinical relevance of ALT, at a fundamental level, the mechanisms of ALT are not well understood. To address these unmet needs, we will identify chemical probes that target ALT using C-circles as a readout for the ALT phenotype. The power of our approach is that the readout is phenotype specific and the compound libraries we will use include target specific libraries, with a focus on epigenetic and kinase targets, as well as diverse chemical libraries. The target focused libraries will allow a chemical genetics approach towards unraveling ALT mechanisms. We anticipate the discovery of small, drug-like molecules to serve as starting points for the discovery of ALT-cancer cell selective agents and probes. We predict that screening target-focused libraries for an ALT-specific phenotype will allow efficient production of chemical probes that will be transformative in unraveling the mechanisms of ALT and allow the evaluation ALT-specific anticancer approaches.